This invention relates to mass spectrometers and their ability to multiplex between simultaneously arriving and discrete sample streams without incurring either sample loss or intra-sample mixing. It concerns itself with the issue of maximizing sample throughput on a mass spectrometer by creating parallel sample introduction and transmission paths, while at the same time ensuring that no mixing of the individual sample streams occurs. In this manner, chemical data are uncompromised in terms of cross-stream contamination, while the overall sample throughput is increased substantially.
This invention is applicable to any mass spectrometer which depends upon batch-wise introduction of samples for performing mass analysis, including but not limited to time-of-flight mass spectrometers (TOF-MS), fourier transform ion cyclotron resonance mass spectrometers (FT-ICR-MS), and three dimensional ion trap mass spectrometers (IT-MS). Time-of-flight mass spectrometers are best suited to exploit this parallel introduction invention because of their inherent ability to process discrete samples on a millisecond time basis or faster. While FT-ICR-MS and IT-MS systems require greater periods of time to acquire high quality mass spectrometric data, these systems could also make use of this invention to improve sample throughput. Commercial FT-ICR-MS systems are currently capable of generating mass spectra at a rate of approximately 50 Hz. While several orders of magnitude lower than TOF-MS systems, this acquisition rate would still permit use of the invention with multiple sample streams, given that these streams could be sampled frequently enough to reflect any temporally dynamic sample concentrations present.
This invention is applicable to any mass spectrometer with an external ion source, and is particularly useful when this ion source produces analytically important ions continuously over extended periods of time. Examples of external ion sources which can produce ions continuously include electrospray ionization (ES) and atmospheric pressure chemical ionization (APCI), both of which may be coupled to liquid chromatography (LC) in order to first temporally separate different species prior to MS interrogation. When coupled to LC or other chemical separation instruments, ES and APCI ion sources generate ions from a temporally dynamic stream of analyte molecules, ranging in duration from seconds (for very fast separations) to several hours (for very long separations).
A fundamental principle of time-of-flight mass spectrometry is the extraction of a closely packed ensemble of ions formed at time zero. These discrete ensembles of isoenergetic and spatially coherent ions are accelerated from an extraction region and into a field free flight tube for longitudinal separation based upon their different (constant) velocities and hence mass-to-charge ratios. Ions created outside the extraction region may be injected into the extraction region, such as from an atmospheric pressure ion source or glow discharge source. Alternately, ions may be created within the extraction region from neutral molecules, for instance by using a pulsed beam of photons, electrons or ions. In either case, only those ions that are in the extraction region at the moment the starting pulse is applied are analytically useful, as only these ions will be imparted with the proper energy to be detected and properly characterized after field-free flight.
Given this constraint, the direct coupling of a continuously operating ion source to a time-of-flight mass spectrometer suffers from an inefficient use of the ions created. While one may apply start pulses to the time-of-flight mass spectrometer at frequencies which match the characteristic time required to re-fill the extraction region from an external supply of ions, duty cycles may still be far from unity under certain conditions.
A solution to this mismatch caused by interfacing a continuous ion source and a batch processing method such as time-of-flight mass spectrometry has been described by Dresch et.al. (1996). In order to make use of the greatest fraction of ions generated as possible, a multipole ion guide is inserted at the appropriate location between the ion source and the extraction region to store ions between consecutive start pulses. Owing to the fact that it is a two dimensional device spanning multiple pumping stages, this device can deliver ions to the extraction region either as a continuously transmitting ion guide or as a pulsed two dimensional ion trap. In contrast to three dimensional ion traps described by Lubman (ref) and Douglas (ref), this two dimensional ion trap can hold a far greater number of ions within its volume before reaching an experimentally observed critical density. Critical density is characterized in practice by the observation of mass spectral signals which may be reduced in amplitude, or different due to catastrophic ion fragmentation, or improperly focussed at the detector due to greater internal energies, or some combination of the above. For a given flux of ions being delivered from an external ion source, the higher charge capacity of this two dimensional ion trap allows storage of ions for more time. This is of the utmost importance to the present invention in affording adequate time for sequentially introducing multiple independent samples through a single time-of-flight mass analyzer without loss of information on the chromatographic timescale.
Ionization methods such as electrospray and atmospheric pressure chemical ionization are utilized regularly to ionize liquid samples containing non-volatile compounds of interest, including but not limited to peptides, proteins, pharmaceutical compounds and metabolites.
The sensitivity, specificity and selectivity of API-MS have made it an essential research tool in the life sciences and pharmaceutical development, in which the analytical performance of API-MS systems has most often been categorized in terms of limits of detection, mass resolving power, mass accuracy, and mass-to-charge range. Previously, little if any regard was paid to issues relating to automation.
Spurred on over the last several years by pharmaceutical development methods, strictly analytical performance metrics have been joined by automation metrics.
Automation of analytical tests such as API-MS afford one or more advantages over manual operation, including:                Reduced labor        Reduced expertise of labor        Higher sample throughput        Better utilization of capital instruments        Better analytical reproducibility (as measured by the relative standard deviations from sample to sample)As an example, the automation of API-MS now allows previously untenable sample sizes to be more rapidly analyzed, thereby supporting technologies such as combinatorial chemistry which require very large sample sizes to isolate a compound of interest.        
As a result, there have been considerable advances in automating the operation and data collection of API-MS instruments both at the hardware and especially the software levels. The latter case is best exemplified by the introduction of Open Access standards for non-expert users. The former case is best illustrated by the introduction of multiple injector autoinjectors such as the Gilson 215 instrument (Madison, Wis.). What has been lacking are the means to accelerate the throughput
Within the last several years, there has been increasing interest in coupling these continuous ionization methods to time-of-flight mass spectrometry in order to achieve certain performance characteristics which would be otherwise unattainable. These include but are not limited to high mass accuracy, high mass-to-charge detection, quasi-simultaneous detection of the entire mass-to-charge domain, high pulse rates, high sensitivity, and fewer tuning requirements than scanning type mass analyzers.
Collectively, these features make time-of-flight mass spectrometers ideally suited as detectors for temporally changing sample streams. Moreover, the ability to couple liquid separation systems directly to atmospheric pressure ionization sources such as electrospray ionization and atmospheric pressure chemical ionization allows for on-line processing of these separations without the need to collect chromatographic or electrophoretic fractions for off-line processing. In fact, the sampling rate of atmospheric pressure ionization time-of-flight mass spectrometers with ideal data system architectures can generate complete mass spectra with adequate ion statistics in far less than 1 second. This speed of acquisition allows faster liquid separation protocols to be designed and implemented which slower, scanning types of mass spectrometers could not record with adequate chromatographic fidelity.
The desire to introduce multiple samples into a single mass analyzer stems from a combination of factors. Technically, time-of-flight mass spectrometers are fast enough in “scanning” a useful mass range that multiple samples can be completely characterized even when these samples are themselves temporally dynamic (as in the case of a liquid chromatogram). For instance, the vast majority of liquid samples separated by reversed phase chromatography will exhibit LC peak widths on the order of several seconds or more. This is ample time for a single TOF-MS to mass analyze several samples, given its ability to form complete mass spectra in as little as 100 microseconds or less.
This multiplexing capability is inviting for those who wish to (a) achieve higher capacity utilization, (b) lower capital costs, (c) shrink total required laboratory space, (d) centralize data handling and (e) minimize hardware maintenance.
There are a number of important works which define the state of the art as it relates to this patent application. These works involve the development of plural ions, parallel mass-spectrometers, and ion storage using two dimensional ion traps. The use of plural ion beams in either single or parallel mass spectrometer has been demonstrated by a number of inventors and for a number of distinctly different reasons. Green in U.S. Pat. No. 3,740,551 demonstrated parallel mass separation and detection of different ion beams simultaneously, principally as a means of performing both high and low resolution mass spectral scans on magnetic sector type instruments. These ion beams could originate from either a single chemical sample or from a sample and a reference compound which was used to calibrate the mass scale of the instrument. In. U.S. Pat. No. 3,831,026 Powers taught the use of a time division multiplexing apparatus, which sampled alternate ion beams for mass separation and detection in an interleaved fashion. This multiplexing apparatus consisted of either a pair of plates at controlled voltages or a continuously transmitting hexapole ion optic. By overtly controlling the portion of time that each ion beam was sampled, relative intensities of the two beams could be better managed for greatest analytical utility. Chang was among the first to recognize the utility of plural beams and parallel mass spectrometers in analyzing temporally dynamic samples from either gas chromatography (GC) or liquid chromatography (LC) in U.S. Pat. No. 4,507,555. Like the aforementioned inventors, parallelism was sought as a means of extracting different types of mass spectrometric data from a single sample, especially in circumstances when rapidly eluting compounds made it difficult or impossible for a slow scanning quadrupole MS to keep pace. One quadrupole was used to monitor a single target mass-to-charge of interest, as well as to trigger full mass range acquisitions by a second quadrupole should the target ion appear. This improved detectability over full mass range survey scans by a factor of 100. Using time-of-flight as the preferred mass separation scheme, Dowel in U.S. Pat. No. 5,331,158 demonstrated the ability to achieve 100% duty cycle of a flight tube (not an individual chemical sample) by injecting ion packets from multiple electron impact ion sources in rapid succession to one another.
Several important patents have been issued in the area of two dimensional ion guides and ion traps, all of which teach important aspects of the science which underpin this patent application. Douglas in U.S. Pat. No. 5,179,278 taught that two dimensional multipole ion guides were highly effective devices for trapping and storing off-cycle ions until a three dimensional ion trap mass spectrometer had completed its analysis of the previous ion bunch. Both pre-selection and collisional cooling of the stored ions were described as advantageous features. Bier in U.S. Pat. No. 5,420,425 furthered this argument by demonstrating the relative analytical advantages of two dimensional ion traps in terms of their storage capacity, circumventing the charge limitations which less stretched ion traps necessarily suffer due to space charge constraints. Both Whitehouse in U.S. Pat. No. 5,652,427 and Dresch in U.S. Pat. No. 5,689,111 describe the use of a multistage two dimensional ion guide as an appropriate ion storage device to feed batch-wise mass spectrometers, including time-of-flight, ion trap and Fourier Transform Ion Cyclotron Resonance type systems. These patents taught the use of enhanced collisional cooling by close coupling a multipole ion guide to the free jet expansion of an atmospheric pressure ionization source. In this way, ions could more effectively be captured while still experiencing viscous forces in the high pressure region of an atmospheric pressure ion source. After capture, their cooling and transport to a much lower pressure region would ensure a much more monoenergetic ion beam which was better suited for injection into energy sensitive MS systems, especially TOF-MS. Franzen in U.S. Pat. No. 5,763,878 extends the multipole ion trap functionality by both trapping ions within the device and using it as the ion source of an orthogonal TOF-MS. Most recently, in U.S. Pat. No. 5,811,800, Franzen generates bunches of stored ions from an atmospheric pressure ion source using RF coils, this time for the purpose of feeding a three dimensional ion trap MS system.
The ability to introduce different samples from different separation systems into a single time-of-flight mass spectrometer was recently introduced by Micromass, Inc. In this design as many as four different liquid streams are multiplexed, with sample selection occurring at atmospheric pressure. This concept is commercially advantageous insofar as it makes use of a standard LC-TOF-MS, requiring no modification of the vacuum system or ion optics to work. However, since all four liquid streams flow continuously, the selection of any one stream necessarily imposes a duty cycle limit dictated by the number of streams sampled. For those streams which are “off-cycle” (i.e. not sampled) any analytical information contained in the off-cycle portions of those liquid streams is lost and can not be recovered. For a large number of applications currently in practice involving high concentrations of synthetically derived small organic libraries, analytical sensitivity is not of paramount concern. Nevertheless, this approach is analytically disadvantageous in circumstances in which sample amounts or concentrations are especially low. Proteomics, including both general molecule characterization as well as peptide sequencing, is a critically important field for which analytical sensitivity is paramount, especially in applications being reduced to nanoscale dimensions for both separation processes (“lab-on-a-chip”) and mass spectrometry (nanoelectrospray).
The present invention arises from the need to mass spectrometrically characterize larger numbers of distinct samples than is currently possible, but without requiring multiple independent mass spectrometers. This analytical need is driven in large part by the adoption of combinatorial chemistry methods by pharmaceutical researchers, who today are the largest and one of the fastest growing segments of the mass spectrometry market worldwide (Strategic Directions International, 1996). Due to this shift towards combinatorial chemistry and away from slower, rational drug design programs, the number of compounds which are being regularly generated and which require positive identification via mass spectrometric analysis has risen dramatically (Doyle, 1995). This trend is expected to continue for years to come (Hail, 1998).
In the field of functional genomics, the ability to identify and characterize gene products (proteins) with vanishingly small amounts of material using mass spectrometry will be essential. Standard separation tools in existence today, including two dimensional electrophoresis, can both separate and detect proteins in amounts far below the detection limits of any mass spectrometer (Ref). While more abundant proteins are easily detected, a large portion of all the proteins contained in mammalian cells exist in copy numbers below the present day capabilities of dedicated, research grade mass spectrometers. Since many of these low abundance proteins are likely to have important regulatory functions in cells, their efficient detection using appropriate staining techniques and their subsequent digestion and analysis using mass spectrometry is vital. (Herbert, Proteome Research: New Frontiers in Functional Genomics). This need is exacerbated by the fact that the entire proteome complement of any organism is a function of age, heredity, wellness, and environmental conditions. Such a dynamic system requires analytical tools which can monitor an organism at various stages of its lifetime. This scarcity of sample will limit the future effectiveness of “lossy multiplexing”, i.e. the use of multiple sample streams multiplexed to a single mass spectrometer with duty cycle limits.
Briefly, syntheses of combinatorially created compounds with potential therapeutic value are carried out using small sets of related starting materials. These sets cover the physical chemical parameters that are required to optimize the properties associated with a pharmaceutical agent, such as good oral bioavailability and in vivo stability. The library or array which results from all possible combinations of these starting materials may be very large in an attempt to cover an appropriate property space, ranging in size from several hundred to several hundred thousand distinct compounds. The complete library or some portion of it which meets certain preliminary screening criteria (the presence or absence of a fluorescence signal, for example) may require complete chemical characterization, usually by mass spectrometry. Because each of the nominal library constituents may be a mixture of the intended product, side-products, reactants, and impurities from various sources, mass spectrometry may be employed in conjunction with a separation method such as liquid chromatography (LC-MS) to separate in time these various components. By separating the individual components within a reaction volume, components elute separately into the ionization source and MS system, generating a mass chromatogram of total ion current versus time. This both simplifies analysis of the data and optimizes the response of the MS system for each constituent by maximizing the ionization efficiency (i.e. minimizing charge competition).
While the chemical specificity of an LC-MS system is greater than using an MS system in the absence of liquid chromatography, there is a time penalty associated with performing an LC separation, reducing the highest achievable sample throughput. The alternative and faster method of analyzing individual liquid samples is by flow injection analysis MS (FIA-MS), infusing liquid samples directly without chromatographic separation.
While the maximum rate at which samples can be sequentially analyzed using either FIA-MS or an LC-MS varies depending upon the specific protocol being followed, in general FIA-MS typically requires between tens of seconds and a minute per sample, depending upon the specific autoinjector hardware being used and the stringency of the inter-sample rinsing. Users in high throughput settings have demonstrated the ability to analyze as many as 1000 samples per mass spectrometer per day in this manner. The primary drawback to this approach is the aforementioned uncertainty in ionization efficiency in the presence of possible impurities. In instances in which the mass spectrometric response is being used as an indicator of the presence or absence of an expected product, the quality of the mass spectrometric data are vital in judging the utility of a particular library compound. Typically one looks for an expected molecular ion of mass M1 to verify synthesis confirmation. If this expected mass is obscured or suppressed by the presence of an impurity with a greater proton affinity of mass M2, then the mass spectrum generated by flow injection MS may not reveal the presence of the target product. However, if the liquid solution containing both of these species is first separated by liquid chromatography or some other appropriate separation which can partition the compounds based upon their physical or chemical properties, then the resultant mass spectra may likely reveal the presence of each of these constituents. In the LC-MS mode, protocols specifically designed for rapid separation of small molecules typically require between 5 and 15 minutes, an improvement over traditional 30–60 minutes gradients used before the advent of high throughput screening but still orders of magnitude slower than other non-mass spectrometric assays. Recently, Banks (1996) demonstrated more rapid separations of complex mixtures in reversed phase LC-MS using both normal bore (4.6 mm ID) and microbore (320 mm ID) columns packed with small uniform spheres of non-porous silica. Separations of 2–3 minutes were typical, demonstrating both high throughput and very high chromatographic resolution. These faster runs were specifically designed to exploit the ability of a time-of-flight mass spectrometer to handle very high data rates. In practice, the compression of chemical separations and the sub-second generation of mass chromatograms by time-of-flight mass spectrometry is the chemical analog of high speed electronic waveform capture, requiring both the means to generate and record events (ions) at the high megahertz to gigahertz frequencies. For this reason, high speed separations coupled to MS have been labelled “burst mode” systems (Banks, 1995). Representative of the current state of the art in high throughput LC-MS, this work clearly shows that radical (order of magnitude or more) improvements in LC-MS throughput, even with specialized chromatographic methods, are not easily obtained when operating in a strictly serial fashion. In order to overcome the sample throughput limitations described here and summarized in Table 1, one of two approaches must be adopted.
First, additional LC-MS instruments, each operating in a serial fashion, could be brought on-line to increase throughput in a strictly linear fashion. This requires a proportionate expenditure of capital and expense funds to purchase and operate multiple machines, as well as requiring multiple computer systems to run the instruments and acquire and analyze data.
Second, multiple separation systems could be coupled in-turn to a single mass analyzer, allowing an LC-MS run to proceed with one LC system while a second LC system is re-equilibrated and a new sample prepared and injected. Such a system has been integrated by the Micromass Division of Waters Corp. for high throughput applications on quadrupole based LC-MS systems. Such an approach is a cost effective means of improving specific sample throughput (in terms of samples per unit time per dollar of realized capital expense), and derives the maximum benefit possible from the relatively expensive mass spectrometer and data system. However, there are two significant limitations. First, the net sample throughput operating two LC systems coupled to a single mass analyzer with a single ion source is far less than two LC-MS systems operating independently. That is, the time savings per sample is approximately equal to that fraction of the time that a single LC system spends re-equilibrating and injecting a new sample onto the column (Figure N).
Third, multiple LC systems could be run in tandem and samples from each be sampled by the MS in turn, using either liquid flow valves or alternating ionization probes to achieve a multiplexing of samples in a single mass analyzer. In the absence of true sample storage, those LC streams which are not being sent to the mass analyzer at any instant in time are being sent to waste. Therefore, this time-slicing approach suffers from the fact that by reducing the duty cycle of each effluent stream, the mass analyzer will be rendered blind to peaks which occur off-cycle. In light of higher speed and higher plate count methods now coming into wider practice, there would be an unreasonably high risk of sending to waste complete peaks which would escape mass spectrometric detection.
The desire to accommodate multiple samples simultaneously in order to achieve higher sample throughput stems in large measure from the growth of combinatorial chemistry. The Biotage Corp. of Charlottesville, Va. produces a product called Parallex HPLC, intended to allow four samples to be chromatographically separated simultaneously. In order to interface these four separate and discrete liquid streams to a mass spectrometer currently, the four streams are routed through a rotary valve which serially introduces each of the four streams to a mass spectrometer's ionization source. In order to prevent stream-to-stream mixing, a bolus of make-up solvent (a “blank”) is introduced into the flow in between consecutive analytical samples. For four separate liquid streams represented by A, B, C, and D, and the make-up solvent represented by S, the sequence of sample delivery to the mass spectrometer will be ASBSCSDSASBSCSDSASBSCSDS . . . . This necessarily implies that the maximum duty cycle achievable for any one of the liquid streams is limited to the portion of time it is actively being sampled, which is one-eight of the total experiment time or 12.5%. For the other 87.5% of the time, those streams which are “offcycle” are not accumulated, but rather are discarded as waste. The time interval required to sample all four liquid streams is on the order of 1 Hz. There are two limitations in coupling such a system to mass spectrometry in order to achieve higher sample throughput. One difficulty is the immediate loss in sensitivity due to the duty cycle limit. Moreover, multiplexing the samples in the liquid phase exacerbates this problem due to the need to introduce inter-sample blanks. The second difficulty is the inability of the multiplexer to select any given liquid stream at a rate greater than 1 or several Hz. Driven by the need to analyze samples ever faster, the clear trend in chromatography is towards faster, higher resolution separations (Ooms). In many cases, separation protocols are now being developed which require only several minutes even for complex mixtures, with eluants exhibiting peak widths of several seconds or less. In instances such as this, mass spectrometric sampling of individual chromatographs at one or several Hz will be inadequate to recreate with any acceptable fidelity the underlying separation. In practice, it is desirable and in many cases required to sample such chromatographs at a rate far higher than the typical elution time of a peak. Typically, sampling the chromatograph at a rate 10 or more times faster than the eluant peak width is acceptable to accurately describe the peak and its fine structure.
The present invention mitigates this time penalty by allowing the simultaneous introduction of more than one liquid separation to the MS system. Furthermore, because of the ion storage feature of the invention, no loss of chromatographic fidelity is incurred, even for chromatograms exhibiting narrow peak widths. This is especially advantageous since high throughput screening applications favor separation systems which can operate at high linear velocities and/or with high numbers of theoretical plates, both of which lead to narrow peaks which could otherwise elute undetected in the absence of ion storage.
One previously described method switches between multiple liquid streams flowing to a single spray assembly for ionization, consecutively valving to waste all but one of the streams at any instant in time (Coffey ref. Because of valve mechanics, this sample selection process is limited in the highest frequency it can operate at while preserving analytically important reproducibility, and moreover creates temporal gaps in the mass chromatograms of the off-cycle streams which may contain analytically important information. Another previously described method advocates the use of multiple ionization assemblies each delivering its distinct sample stream in sequence to a single vacuum orifice. Gating of the individual ionization assemblies may occur by modulation of a combination of: (1) electric potential to the spray probe; (2) pneumatic gas pressure and flow to the spray probe; (3) gas pressure, flow and orientation to the countercurrent bath gas; and/or alignment and positioning of the individual spray probes with respect to the vacuum orifice.
Making use of the high sampling rate of the time-of-flight electronics and the storage capabilities of two dimensional multipole ion traps. In this manner, more than one liquid handling system can continuously infuse its effluent or other the simultaneous introduction of multiple sample streams to multiple atmospheric pressure ionization spray assemblies.